The acidic amino acid, glutamate, is the predominant excitatory neurotransmitter in the mammalian central nervous system. Although there are millimolar concentrations of this excitatory amino acid (EAA) in the brain, extracellular concentrations are maintained in the low micromolar range to facilitate crisp synaptic transmission and to prevent excessive activation of receptors that can kill neurons (or other cells that express glutamate receptors). A family of Na^-dependent high- affinity glutamate transporters is responsible for the regulation and clearance of extracellular EAAs. This project represents a collaborative effort between two laboratories that have contributed to our understanding of the biochemical, pharmacologic, anatomic, and pathophysiologic properties of these transporters. In this competitive renewal, we propose to continue focusing on one of the astroglial glutamate transporters, called GLT-1 or EAAT2. There is substantial evidence to suggest that this transporter mediates the largest percentage of glutamate uptake activity in the forebrain. The expression (mRNA and protein) of GLT-1/EAAT2 increases during synaptogenesis in vivo and is induced in astrocytes in vitro by the presence of neurons. Lesions of projection neurons in vivo results in decreased GLT-1/EAAT2 expression in target areas. In addition, several acute and chronic neurodegenerative diseases (or animal models of these diseases) are accompanied by decreased expression of GLT-1/EAAT2. Based on our prior studies and our preliminary data, we propose to identify signals/transcription factors and promoter elements that contribute to basal and 'neuron- dependent' induction of GLT-1/EAAT2 (Aims I & II). In pur preliminary studies, we have also identified fragments of the GLT-1/EAAT2 5' non-coding region that contain elements that suppress transcription. These elements will also be characterized (Aims I & II). As we identify elements that are required for induction/suppression of GLT-1 expression, these elements will be introduced into models of neurologic insults with the goal of determining if the presence damaged/dying neurons results in decreased promoter activation or results in active suppression of GLT-1 transcription. With this approach, we hope to develop an understanding of the mechanisms that regulate the expression of the predominant protein that limits excitability in the normal nervous system and to develop an understanding of the factors that may contribute to the loss of this protein in both acute and chronic neurodegenerative diseases. Children's Hospital of Philadelphia, Philadelphia, Pennsylvania The Johns Hopkins School of Medicine, Baltimore, Maryland PHS 398 (Rev.09/04) Page 2 Form Page2 Principal Investigator/Program Director (Last, First, Middle): Robinson, Michael B. KEY PERSONNEL. See instructions. Use continuation pages as neededto provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Michael B. Robinson, Ph.D. Michael_Robinson Children's Hospital PI Jeffrey Rothstein, MD PhD JRothstl Johns Hopkins Co-Pi OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Beverly Davidson, Ph.D. University of Iowa Consultant Shelley Russek, Ph.D. Boston University Consultant John Wolfe, V.M.D./Ph.D. CHOP/University of Pennsylvania Consultant Human Embryonic Stem Cells X No CH Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell llne(s) from the following list: http://stemcells.nih.gov/reqistrv/index.asp. Usecontinuationpagesasneeded. If a specific line cannot be referenced at this time, include a statement that onefrom the Registry will be used. Cell Line Disclosure Permission Statement. Applicable toSBIR/STTR Only. See SBIR/STTR instructions. 0 Yes O No PHS 398 (Rev.09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Robinson, Michael B. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page i_ Description,